Interpretive Summary: Plants in the legume family, which includes alfalfa, peas, and beans, form an important symbiotic association with soil bacteria in root nodules in which the bacteria transform atmospheric nitrogen gas into ammonia. The plants utilize the ammonia to produce amino acids used in synthesizing proteins. In barrel medic, a plant closely related to alfalfa, the predominant proteins in root nodules are nodule cysteine-rich proteins (NCRs), which are similar to defensins, proteins that have antimicrobial activity. However, the function of NCRs is unknown. This study was undertaken to gain an understanding of when NCRs are produced, what signals regulate their production, and to test several NCRs for inhibiting pathogens. We found that the NCRs in nodules can be separated into two groups. Members of one group are produced early in nodule formation as bacteria enter the nodule, and members of the second group are produced once bacteria initiate nitrogen fixation. Thus, NCR production is regulated by bacterial number or volume occupied by bacteria. We also identified unique DNA sequences in NCR genes that are necessary for production of NCR messenger RNA in nodules. Knocking out expression of several predominant NCRs did not affect nodule formation or function nor did it affect disease resistance, indicating a redundant function of the many NCRs found in nodules. Our results suggest that NCRs may have a role in protecting nodules from the other organisms in soil and act as signal molecules in plant development. This work enhanced understanding of a large group of plant proteins that have potential for increasing plant productivity through nutrient acquisition and in defense against pathogenic microorganisms.

Technical Abstract:
Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the largest gene families expressed in the nodules of the model legume Medicago truncatula is the nodule cysteine-rich (NCR) group of defensin-like (DEFL) genes. We used a custom Affymetrix oligonucleotide microarray to examine the expression changes of 566 NCRs at different stages of nodule development. Additionally, rhizobial mutants were used to understand the importance of the rhizobial components in induction of NCRs. Expression of early NCRs was detected during the initial infection of rhizobia in nodules and expression continued as nodules became mature. Late NCRs were induced concomitantly with bacteroid development in the nodules. The induction of early and late NCRs was correlated with the number and morphology of rhizobia in the nodule. Conserved 41 to 50 bp motifs identified in the upstream 1,000 bp promoter regions of NCRs were required for promoter activity. These cis-element motifs were found to be unique to the NCR family among all annotated genes in the M. truncatula genome, although they did contain sub-regions with clear similarity to known regulatory motifs involved in nodule-specific expression and temporal gene regulation.